A comparative study of fungicidal activities of voriconazole and amphotericin B against hyphae of Aspergillus fumigatus

Abstract

To study the in vitro fungicidal activity of voriconazole against hyphae of Aspergillus fumigatus and compare the results with those obtained for the known fungicidal drug amphotericin B. A. fumigatus mycelia were grown on Sabouraud dextrose agar and in peptone yeast extract glucose broth until the cultures reached a mid-logarithmic growth phase. The fungicidal activities of voriconazole and amphotericin B against actively growing hyphae of A. fumigatus were examined by a kill-curve experiment and a fungal cell viability test. For the kill-curve study, the drug-treated hyphae were washed, homogenized and resuspended in 1 mL of sterile water, diluted 10-1000 fold and aliquots of 0.1 mL were spread on Sabouraud dextrose agar and allowed to grow for 48 h at 35 degrees C. The cfu were determined and plotted against drug concentrations for each time of exposure to obtain the kill curve. The viability of drug-treated A. fumigatus hyphae was determined by their ability to reduce tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. Exposure of A. fumigatus hyphae to several concentrations (1-16 mg/L) of voriconazole or amphotericin B for various time intervals killed the hyphae in a time- and drug concentration-dependent manner. Voriconazole at 1 mg/L killed >95% of the hyphae grown on Sabouraud dextrose agar after 48 h of exposure, whereas amphotericin B at the same concentration killed approximately 70% of the hyphae after exposure for the same duration. Approximately 99% killing of hyphae grown in peptone yeast extract glucose broth was obtained for voriconazole at 1 mg/L after 48 h of exposure, whereas amphotericin B at 1 mg/L yielded approximately 82% killing after 48 h. The fungal cell viability test by tetrazolium reduction assay showed that mycelia exposed to > or =1 mg/L (Sabouraud dextrose agar blocks) and > or =2 mg/L (broth cultures) of voriconazole for 48 h completely failed to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. At low concentrations (1-2 mg/L) amphotericin B had no detectable effect on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction by drug-treated mycelia, whereas mycelia treated with 16 mg/L for 48 h showed approximately 50% inhibition of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction compared with the control. Voriconazole possesses excellent fungicidal activity against actively growing hyphae of A. fumigatus. A comparison of results with those obtained for the known fungicidal drug amphotericin B shows that, in peptone yeast extract glucose broth, voriconazole has superior fungicidal activity against A. fumigatus hyphae compared with that of amphotericin B.